Colin A et al. (2016), Triggering signaling pathways using F-actin sel...

XB-ART-55050

Sci Rep 2016 Oct 04;6:34657. doi: 10.1038/srep34657.

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Triggering signaling pathways using F-actin self-organization.

Colin A , Bonnemay L , Gayrard C , Gautier J , Gueroui Z .

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The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.

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Species referenced: Xenopus laevis
Genes referenced: mapre1 nps ran utrn

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	Figure 1. Membrane-free confinement mediated by F-actin self-organization to localize and trigger signaling pathways.(a) The confinement of egg extracts within oil droplets leads to the spontaneous generation of a centripetal F-actin flow that eventually forms a contractile ring-like structure. F-actin flow conveys cytoplasmic materials that are trapped by the filament meshwork. Actin microfilaments were labeled with Utr-GFP. Scale bar is 10â€‰Î¼m. (b) Schematic of the concept of signaling localization and switch: F-actin self-organization powered the active compartmentalization of nanoparticles operating as signaling platforms or the scaffolding of signaling proteins by F-actin polymers. Starting from a homogeneous distribution of signaling proteins, the generation of the F-actin flow drives the concentration enhancement of regulatory elements that could trigger signal pathway. (c) The activity of Ran GTPase is characterized by a non-linear response following a sigmoidal response with the concentration (ultrasensitive switch). The nucleation of microtubules is triggered above a concentration threshold of Ran. Our hypothesis is that the F-actin self-organization into a contractile meshwork will trigger the Ran signaling pathway and the nucleation of microtubules.
	Figure 2. Characteristics of the membrane-free compartmentalization mediated by F-actin flow.(a) F-actin dynamics transport nanoparticles in a size-dependent manner. Accumulation of nanoparticles of different sizes (50â€‰nm, 120â€‰nm, and 300â€‰nm) shows that only nanoparticles above a diameter of 100â€‰nm are transported by F-actin flow. (b) Time-dependent accumulation of 120â€‰nm fluorescent nanoparticles. After 6â€‰minutes, almost all the nanoparticles are gathered at the center of the F-actin ring. (c) Confocal observations of F-actin ring-like structures show that microfilaments encompass cytoplasmic materials stained with membrane marker. Slices of a z-stack; images are 8â€‰Î¼m apart (from 5 to 45â€‰Î¼m in the droplet). (d) Left: Displacement field of 300â€‰nm-nanoparticles reveals that their motions are highly directed towards the F-actin ring. Right: Angular distribution of tracked trajectories with respect to the actin ring position. (e) Estimation of the cytoplasmic diffusion coefficients in cell extracts incompetent for F-actin organization, and contractile cell extracts at early stage and late stage of F-actin ring organization. Mean and standard deviation are plotted. Scale bars are 10â€‰Î¼m.
	Figure 3. Characterization of asters nucleated with RanQ69L in an F-actin intact cell extract.(a) Microtubule asters nucleated with 8â€‰Î¼M of RanQ69L. The microtubules are localized and confined within F-actin ring. mCherry-labeled RanQ69L observation indicates an homogenous distribution of Ran within the droplet. (b) Two types of morphologies are observed when microtubule nucleation is triggered in presence of F-actin: asymmetric asters localized inside the F-actin ring and asymmetric asters localized outside the F-actin ring. In absence of F-actin we observed a radial aster. F-actin was labeled with Utr-GFP and fluorescent (TRITC) tubulin was used to visualize microtubules. Scale bars are 10â€‰Î¼m.
	Figure 4. Signaling switch triggered by F-actin flow and by the active confinement of Ran-nanoparticles.(a) Estimation of the concentration enhancement driven by F-actin self-organization by quantifying the lysotracker spatial distribution along the droplet diameter. (b) Schematic of the proof-of-concept experiment. GTPase RanQ69L are grafted on nanoparticles and the complexes are dispersed within cell extract droplets at a concentration level below the threshold required for microtubule growth. The F-actin flow conveys and confines the Ran-nanoparticles within the F-actin ring-structure to eventually induce a concentration enhancement of Ran in a restricted area, which may lead to activate microtubule nucleation.(c) Ran-nanoparticles control microtubule assembly with an activity characterized by a sigmoidal concentration dependency. (d) Confocal observation of a microtubule-aster formation induced by the active confinement of Ran-NPs (initially added at a concentration under nucleation threshold, 500â€‰nM). Microtubules are stained with rhodamine-labeled tubulin, and F-actin with Utr-GFP. (e) Quantification of the efficiency of the signaling switch: percentage of droplets containing an aster in presence or absence of F-actin flow. The box plot shows the median (central mark), the 25th and 75th percentiles (edges of the box); the whiskers extend to the most extreme data points that are not considered as outliers.
	Figure 5. Signaling switch mediated by F-actin polymeric scaffolds.(a) Principle of the proof-of-concept experiment examining how F-actin could act as polymeric scaffold hubs to recruit regulatory proteins. RanQ69L is fused to the CH domain of utrophin to target F-actin filaments. Contraction and F-actin density increase may drive the concentration increase of Utr-Ran in a restricted area to trigger microtubule polymerization. (b) Evaluation of the fold increase in density of F-actin meshwork after contraction. (c) Co-localization of Utrophin-emGFP-RanQ69L (green) with F-actin meshwork (Alexa-568 Phalloidin, red). (d) Utr-RanQ69L induces microtubule assembly in confined extracts with a sigmoidal concentration dependency. (e) Confocal observation of microtubule formation induced by Utr-RanQ69L under nucleation threshold (500â€‰nM) in confined extract. Microtubules are stained with rhodamine-labeled tubulin, and F-actin with Utr-GFP. (f) Quantification of the efficiency of the signaling switch mediated by F-actin flow: number of droplets containing microtubule arrays with F-actin flow and with the inhibition of F-actin flow. Observations were done with confocal microscopy. (g) Confocal observation of microtubules induced by GFP-Utr-Ran under nucleation threshold (500â€‰nM). Microtubules are stained with rhodamine-labeled tubulin. (h) Left: typical kymograph extracted from steric trapping occurs within 5â€‰minutes (Fig. 2b and Movie 3. Microtubules are labeled with EB1-GFP. Right: quantitative analysis of the growth rate extracted from the kymographs. Growth rates were measured for microtubules assembled using Utr-RanQ69L that are recruited to F-actin scaffolds (initial concentration at 500â€‰nM) and using RanQ69L (6â€‰Î¼M). Mean and standard deviation are plotted. The measured growth rates are similar between these two pathways for growth (Utr-RanQ69L and RanQ69L). Scale bars are 10â€‰Î¼m.

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